IL-6 INDUCTION OF PROTEIN-DNA COMPLEXES VIA A NOVEL REGULATORY REGIONOF THE INDUCIBLE NITRIC-OXIDE SYNTHASE GENE PROMOTER - ROLE OF OCTAMER BINDING-PROTEINS

Macrophage inducible nitric oxide synthase (iNOS) catalyzes the synthe sis of NO. IL-6-stimulated macrophage differentiation of murine myeloi d Mi cells is accompanied by iNOS gene induction and steady-state mRNA expression, Two regions within the iNOS promoter mediate transcriptio nal responsiveness to LPS and IFN-gamma. Region I contains several ess ential transcription factor binding motifs and promotes responsiveness to LPS, whereas region II potentiates the LPS response by IFN-gamma. Because region I possesses basal promoter activity and directly mediat es iNOS gene activation, we attempted to identify the trans-acting fac tors involved in IL-6-stimulated induction of the murine iNOS gene thr ough this region, Using an electrophoretic mobility shift assay and me thylation interference, we show that IL-6 induced reciprocal changes i n the binding activity of POU family members to the candidate nonconse nsus octamer sequence of region I that correlated, temporally, with iN OS steady-state mRNA expression, Although DNA-protein binding activity of IL-6-stimulated whole-cell extracts also interacted with a radiola beled canonical octamer motif, such DNA-protein complexes were not eli minated in competition assays using consensus nuclear factor KB Or IL- 6 oligonucleotides, Specifically, our studies show that octamer bindin g protein-1-related protein binding activity decreased, while binding of octamer binding protein-2-related proteins increased during differe ntiation. Mutation of the octamer motif disrupted both binding of the IL-6-induced protein-DNA interactions and transcriptional activation t hrough region I, revealing that this motif is absolutely essential for IL-6 induction of iNOS, Thus, differential activation of octamer bind ing transcriptional modulators from the POU family may be a novel mech anism of IL-6-mediated iNOS gene regulation.